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Biomedical subjects

E Wilson

Publications and source records attributed to E Wilson.

At least 19 recordsLinked to original sources
PubMed1992

Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis.

Stimulation of certain receptor tyrosine kinases results in the tyrosine phosphorylation and activation of phospholipase C gamma (PLC gamma), an enzyme that catalyses the hydrolysis of phosphatidylinositol (PtdIns). This hydrolysis generates diacylglycerol and free inositol phosphate, which in turn activate protein kinase C and increase intracellular Ca2+, respectively. PLC gamma physically associates with activated receptor tyrosine kinases, suggesting that it is a substrate for direct phosphorylation by these kinases. Here we report that a fibroblast growth factor (FGF) receptor with a single point mutation at residue 766 replacing tyrosine with phenylalanine fails to associate with PLC gamma in response to FGF. This mutant receptor also failed to mediate PtdIns hydrolysis and Ca2+ mobilization after FGF stimulation. However, the mutant receptor phosphorylated itself and several other cellular proteins, and it mediated mitogenesis in response to FGF. These findings show that a point mutation in the FGF receptor selectively eliminates activation of PLC gamma and that neither Ca2+ mobilization nor PtdIns hydrolysis are required for FGF-induced mitogenesis.

Amino Acid Sequence↗
PubMed1992

Effect of diltiazem on norepinephrine-induced acute left ventricular dysfunction.

The present study has examined the role of diltiazem as a protective agent in a canine model of norepinephrine cardiotoxicity. Effects of diltiazem, 20 micrograms/kg/min x 5 min pretreatment followed by 10 micrograms/kg/min x 90 min, or saline infusion were examined at baseline and 1 h after infusion of norepinephrine, 4 micrograms/kg/min for 90 min, in closed-chest anesthetized dogs. Left ventricular function was assessed by equilibrium radionuclide angiogram and two-dimensional echocardiogram. In 7 saline experiments, hypotension and increased heart rate were observed at 1 h post infusion. In the diltiazem-treated group (n = 7), mean arterial pressure and heart rate were unchanged. In the saline group, left ventricular ejection fraction fell from 0.50 +/- 0.04 to 0.28 +/- 0.04. Left ventricular ejection fraction was unchanged in the diltiazem-treated group: 0.52 +/- 0.03 to 0.55 +/- 0.07. Left ventricular end-diastolic volume was increased at 1 h post infusion in saline controls but not in the diltiazem-treated group. Measurement of fractional shortening from two-dimensional echocardiograms also indicated left ventricular dysfunction in the saline but not diltiazem-treated groups. Left ventricular end-systolic wall stress following norepinephrine infusion was significantly increased in the saline but not diltiazem-treated groups: 122.7 +/- 18.7 vs 67.6 +/- 19.7 g/cm2, respectively. In dogs receiving saline without norepinephrine infusion, no significant changes occurred over the course of the experiment. Histologic examination showed mild contraction band necrosis in saline controls but not in sham saline dogs. Diltiazem showed intermediate histologic evidence of injury, which was not further quantified. This study suggests that effects of norepinephrine on left ventricular function in the canine model of norepinephrine cardiotoxicity may be largely due to increased wall stress following a prolonged increase in afterload. Diltiazem pretreatment afforded significant protection of left ventricular function.

Animals↗
PubMed1992

Labeling and stability of radiolabeled antibody fragments by a direct 99mTc-labeling method.

The in vitro labeling and stability of 99mTc-labeled antibody Fab' fragments prepared by a direct labeling technique were evaluated. Eight antibody fragments derived from murine IgG1 (N = 5), IgG2a (N = 2) and IgG3 (N = 1) isotypes were labeled with a preformed 99mTc-D-glucarate complex. No loss of radioactivity incorporation was observed for all the 99mTc-labeled antibody fragments after 24 h incubation at 37 degrees C. The 99mTc-labeled antibody fragments (IgG1, N = 2; IgG2a, N = 2; IgG3, N = 1) were stable upon challenge with DTPA, EDTA or acidic pH. Furthermore, using the affinity chromatography technique, two of the 99mTc-labeled antibody fragments displayed no loss of immunoreactivity after prolonged incubation in phosphate buffer up to 24 h at 37 degrees C. The bonding between 99mTc and antibody fragments was elucidated by challenging with a diamide ditholate (N2S2) compound. The Fab' with IgG2a isotype displayed tighter binding to 99mTc in comparison to the Fab' from IgG1 and IgG3 isotype in N2S2 challenge and incubation with human plasma. The in vivo biodistribution of five 99mTc-labeled fragments were evaluated in normal mice. In conclusion, the direct labeling method allows stable 99mTc labeling of antibody fragments from three of the major murine isotypes.

Chelating Agents↗
PubMed1992

A recombinant calcitonin receptor independently stimulates 3',5'-cyclic adenosine monophosphate and Ca2+/inositol phosphate signaling pathways.

Calcitonin (CT), a polypeptide hormone, regulates calcium homeostasis by activating surface receptors coupled to stimulation of adenylyl cyclase in bone and kidney cells. CT has also been reported to increase cytoplasmic Ca2+ in osteoclasts and renal tubule cells. Signaling pathways activated by a recombinant porcine renal calcitonin receptor transiently expressed in HEK-293 cells were studied. In cells expressing the recombinant CT receptor, salmon CT stimulated cAMP accumulation (EC50, 0.16 nM) and synthesis of inositol phosphates (IP; EC50, 3.7 nM). Two other recombinant receptors, the m1-muscarinic acetylcholine receptor and the LH receptor, activated synthesis of either IP or cAMP, respectively, but not both. Stable expression of the CT receptor in a CT receptor-deficient cell line, M18, restored the cells' ability to increase cytoplasmic Ca2+ in response to salmon CT. These results show that a single recombinant CT receptor can independently activate effector pathways mediated by cAMP and IP/Ca2+.

Animals↗
PubMed1991

Evaluation of the 323/A3 monoclonal antibody and the use of technetium-99m-labeled 323/A3 Fab' for the detection of pan adenocarcinoma.

The 323/A3 murine monoclonal antibody, initially described as reactive to breast carcinomas, is found by immunohistological analyses to have broad cross reactivity with adenocarcinomas of diverse histologic origin. The 323/A3 antigen is similar to the tumor-associated 17-1A antigen as revealed by immunoblot and cross-competition cell binding studies. We have investigated the potential use of the 323/A3 monoclonal antibody for tumor imaging as a Fab' molecule labeled with 99mTc. In vitro studies demonstrate that 323/A3 Fab' has high affinity (2-3 x 10(9) M-1) with no significant loss of immunoreactivity compared to the intact IgG. In vivo studies demonstrate that 99mTc 323/A3 Fab' can rapidly detect human breast and colon tumor xenografts growing in athymic nude mice. Distinct breast tumor visualization is observed as early as 1 h post intravenous administration with the 99mTc 323/A3 Fab'. Distinct colon tumor visualization is observed by 3 h (the earliest time point imaged). Tumor-to-blood ratios are higher for 99mTc 323/A3 Fab' than with a 99mTc-labeled nonspecific isotype-matched Fab' antibody. These results suggest that 99mTc 323/A3 Fab' can detect 17-1A antigen and may have potential clinical utility for the rapid diagnostic imaging of adenocarcinomas.

Adenocarcinoma↗
PubMed1991

Enhancement of macrophage superoxide anion production by amphotericin B.

Amphotericin B (AmB) appears to have some important immunomodulatory effects, but its mechanism of action has not been explained. We investigated the effects of AmB on activation of human monocyte-derived macrophages. Macrophages cultured in the presence of AmB had an enhanced capacity to produce superoxide anion after stimulation with phorbol myristate acetate. This enhancement was dose dependent within a therapeutic range of AmB levels (0.1 to 3.0 mg/liter). Macrophages cultured in the presence of AmB had enhanced surface expression of Ia antigen; phagocytosis of unopsonized zymosan, opsonized Staphylococcus aureus, or erythrocytes opsonized with C3bi or immunoglobulin G paradoxically appeared to be reduced, but results did not achieve statistical significance. AmB appears to activate macrophages and may do so via direct effects on the plasma membrane.

Amphotericin B↗
PubMed1991

AIDS-related attitudes and knowledge among attenders at a Scottish family planning clinic.

AIDS-related attitudes and knowledge of 1,970 family planning clinic attenders were examined using a self-completion questionnaire. Attitudes towards testing varied depending on the sex, age and marital status of respondents. Knowledge of HIV transmission routes was generally good, although the fact that those most likely to be at risk of infection--the younger respondents and those not currently in steady relationships--were the least well-informed gives some cause for concern. Since family planning consultations are frequently the only contact these young sexually active individuals have with the health service the opportunities for health education on HIV/AIDS afforded by regular attendance at these clinics should be capitalized upon.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Lymphocytic leukemia and lymphosarcoma in a rabbit.

Lymphocytic leukemia and lymphosarcoma were diagnosed in a rabbit with lethargy, emaciation, and pallor. The diagnosis was made on the bases of results of hematologic analysis, cytologic evaluation of a bone marrow specimen, and histologic examination. The lymphosarcoma was identified to be of T-cell origin. Leukemia is rarely diagnosed in rabbits, although lymphosarcoma is fairly common in this species.

Animals↗
PubMed1990

Radiolocalization of human prostate tumor in a mouse subrenal capsule model by monoclonal antibody TURP-27.

The subrenal capsule assay was used to determine if 125I-labeled anti-prostate monoclonal antibody TURP-27 could target human prostate tumor fragments implanted under the renal capsule of normal immunocompetent C57 BL/6 mice. Maximal binding and optimal tumor to non-tumor tissue ratios occurred within 24-48 hours postadministration of 125I-TURP-27. No significant localization was observed in mice bearing TURP-27 antigen-negative human colon tumor tissue implants or with an isotype-matched control monoclonal antibody. These preclinical data suggest that TURP-27 may have clinical application for imaging metastatic prostate tumors and further application in immunoconjugate and/or radiotherapy of prostate cancer.

Animals↗
PubMed1990

Mutant analysis approaches to understanding calcium signal transduction through calmodulin and calmodulin regulated enzymes.

An example set of site-specific mutagenesis studies of calmodulin has been discussed in terms of strategy and how the results can provide insight into the functioning of calmodulin. A set of common examples for the study of calcium binding and enzyme activation were discussed. Essentially, site-specific mutagenesis in these initial studies is a perturbation approach. From these perturbation studies, structural features can be correlated in future studies with function and mechanisms of action proposed. More importantly, the approach allows efficient testing of proposed mechanisms and further probing of the molecular aspects of the signal transduction pathways. Clearly, the key functional feature that must be addressed in future studies is how the calcium binding steps in the mechanism are coupled to the enzyme activation step, which is the final step of the calmodulin-enzyme binding mechanism.

Amino Acid Sequence↗
PubMed1990

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity.

The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.

Amino Acid Sequence↗
PubMed1990

Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection.

The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence. Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically. A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium. Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity. However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin-regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule. Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects.

Amino Acid Sequence↗
PubMed1990

Sedation during spinal anaesthesia: comparison of propofol and midazolam.

Propofol and midazolam were compared in 40 patients undergoing orthopaedic surgery under spinal anaesthesia. An infusion of either 1% propofol or 0.1% midazolam was given at a rate adjusted to maintain a similar level of sedation. The mean time to reach this required level was similar in both groups. Quality and ease of control of sedation were good in all patients. A mean infusion rate of 3.63 mg kg-1 h-1 was required for propofol and 0.26 mg kg-1 h-1 for midazolam. Immediate recovery, as judged by ability to open eyes and recall date of birth, was significantly more rapid following propofol (P less than 0.001). Similarly, restoration of higher mental function was significantly faster following propofol, measured by choice reaction time and critical flicker fusion threshold. Amnesia for the immediate postoperative period was significantly greater after midazolam (P = 0.0001).

Anesthesia Recovery Period↗
PubMed1990

Disk elution method for MICs and MBCs.

A MIC macrodilution broth method in which routine antimicrobial susceptibility testing disks were used as a source of antibiotic was compared with the M7A reference method of the National Committee for Clinical Laboratory Standards for accuracy and reproducibility over five test runs. Gentamicin, piperacillin, and oxacillin were tested by using American Type Culture Collection quality control strains and laboratory isolates for which the MIC of each agent was known. An optimum time for elution of 1 h was first determined by timed elution in broth, confirming levels determined by the TDX method (Abbott, North Chicago, Ill.) for gentamicin and bioassay for piperacillin and oxacillin. MICs determined by the disk elution method demonstrated a reproducibility rate of 97% as well as 97% agreement with the reference method to within +/- 1 dilution; MBCs showed 93% agreement. The disk elution method is reproducible and provides a reliable test system that can be used by any laboratory in which disk susceptibility testing is performed.

Anti-Infective Agents↗
PubMed1990

Aerosolization of recombinant SLPI to augment antineutrophil elastase protection of pulmonary epithelium.

In a variety of lung diseases the respiratory epithelial surface must contend with an increased burden of neutrophil elastase (NE). One candidate for augmenting epithelial anti-NE protection is the secretory leukoprotease inhibitor (SLPI). In vitro evaluation demonstrated that 96 +/- 1% of the recombinant SLPI (rSLPI) molecules were capable of inhibiting NE, with an association rate constant of 7.1 +/- 0.1 X 10(6) M-1.s-1. Evaluation of rSLPI after in vitro and in vivo aerosolization showed that aerosolization did not alter rSLPI. Aerosolization of a single dose of 50 mg rSLPI to sheep resulted in a fourfold increase of the anti-NE capacity in epithelial lining fluid (ELF) at 3 h, with a half-life in ELF of 12 h. After aerosolization some rSLPI appeared in lung lymph. Simultaneous aerosolization of rSLPI and recombinant alpha 1-antitrypsin (rAAT) demonstrated a molar ratio of the concentration in lymph to the concentration in ELF 3 h after the aerosol eightfold higher for rAAT than for rSLPI. Overall, these observations demonstrate that it is feasible to use aerosolized rSLPI to directly augment the anti-NE capacity of the lung, particularly on the pulmonary epithelial surface.

Aerosols↗